In Vitro Tumor Metastasis Model

ABSTRACT

This invention provides a system and methods for modeling tumor metastasis in vitro where primary tumor tissue is cultivated in an orientation and an environment such that the natural composition, three-dimensional organization, and environmental conditions of the tumor can be adjusted. The invention further provides mechanism for inducing tumors to undergo metastatic processes resulting in production of tumor progenitor or stem cells that can be collected, characterized, or used to induce tumors in normal tissue constructs in vitro.

FIELD OF THE INVENTION

This invention relates to the field of cancer biology. Morespecifically, the invention relates to in vitro systems for culturingcancer cells and tissues.

BACKGROUND OF THE INVENTION

The majority of cancer deaths are due to complications from metastasisto distal organs. Developing an in vitro model that is representative ofthe metastasis process will significantly expedite research of themolecular events associated with metastasis and will contribute to thedevelopment of novel therapies that target metastatic processes.

The current standard for metastatic tumor models is represented by invivo models (xenografts) involving mice, rats or other animal speciescommonly employed for medical research. These models suffer from anumber of significant drawbacks including low and unpredictable rates oftumor engraftment and the confounding influences of non-human molecularfactors from the host species, see e.g. Quintana et al., (2008),Efficient tumor formation by single human melanoma cells, Nature456(7222):593. Both in vivo and traditional in vitro models also havethe drawback of losing human stromal cells normally associated with thetumor, see e.g., Montel et al., (2006) Tumor-stromal interactionsreciprocally modulate gene expression patterns during carcinogenesis andmetastasis. Int. J. Cancer, 119:25. There is also difficulty inidentifying and isolating any metastatic cancer stem cells that may bereleased by tumors into the complex milieu of the host animal'scirculatory system, see e.g. Talmadge and Fidler, (2010) AACR CentennialSeries: The Biology of Cancer Metastasis: Historical Perspective, CancerRes; 70(14):5649.

Another reason aspect for switching to in vitro models for studyingmetastasis is ethical considerations. If good alternatives to animalmodels are available they are preferred. The European Union Directive2010/63/EU on the protection of animals used for scientific purposesstates that it is: “essential, both on moral and scientific grounds, toensure that each use of an animal is carefully evaluated as to thescientific or educational validity, usefulness and relevance of theexpected result of that use. The likely harm to the animal should bebalanced against the expected benefits of the project.” Furthermore,there are many documented cases were animal models fail to accuratelypredict medical outcomes in humans. See e.g., Knight, A. (2007)Systematic reviews of animal experiments demonstrate poor human clinicaland toxicological utility. Altern. Lab. Anim. 35:641; Gura T. (1997)Systems for identifying new drugs are often faulty. Science 278:1041.

Traditional in vitro methods for studying metastasis typically involvethe use of tumor cell lines cultivated in two-dimensional systems withlittle ability to simulate gradients of nutrients, gases, and otherfactors present in the natural in vivo environment. These cellline-based systems lack the mixed cell populations, the naturalthree-dimensional arrangements of cells, or the variedmicro-environments present in tumors in vivo. As a result of thelimitations described above, both in vivo and in vitro methods currentlyin use lack “biorelevance” and are poorly predictive of natural tumorbehavior.

SUMMARY OF THE INVENTION

This invention provides a culture system and methods for modeling tumormetastasis in vitro where the tumor tissue is cultivated in anorientation and in an environment such that the natural composition,three-dimensional organization, and environmental conditions of thetumor can be simulated and controlled. The invention further providesmechanisms for inducing tumors to undergo metastatic processes resultingin production of tumor progenitor or stem cells that can be collected,characterized, or used to induce tumors in normal tissue constructs invitro.

In some embodiments, the invention is a combination of athree-dimensional bioreactor and one or more tumor cells in which thesystem parameters are such that the tumor cells maintain their normalmetastatic potential. In variations of this embodiment, the bioreactorcomprises a cell-supporting but cell-permeable matrix separating atleast two fluid chambers with fluid flowing therethrough and at leastone gas chamber connected to each of the fluid chambers and the tumorcells have been introduced into one of the at least two fluid chamberscontaining suitable nutrient medium and gas sufficient to sustain tumorgrowth. In further variations of this embodiment, the metastatic cellsinclude cells having characteristics of circulating tumor cells andcirculating tumor progenitor cells including the presence of one or moreof the following: EpCAM, cytokeratins (CK) 5, 7, 18 and 19, IGF-1R,BCL2, HER2, EphB4, CA19-9, CEA, CD133, MUC1, Survivin, PTEN, CD44v6,N-cadherin, and FAP (Seprase).

In another embodiment, the invention is a method of generatingmetastatic tumor cells in vitro comprising: introducing one or moretumor cells into a bioreactor; providing to the bioreactor fluid culturemedia and gas composition supportive of growth of the tumor cells;incubating the bioreactor under conditions and for a time sufficient forthe tumor cells to proliferate and produce metastatic cells; andcollecting metastatic tumor cells. In variations of this embodiment, thebioreactor comprises a cell-supporting but cell-permeable matrixseparating at least two fluid chambers with fluid flowing therethroughand at least one gas chamber connected to each of the fluid chambers. Infurther variations of this embodiment, the tumor cells are introducedinto the first fluid chamber and metastatic tumor cells are collectedfrom among cells that have migrated into the second chamber.

In yet another embodiment, the invention is a method of manipulating aculture of tumor cells in a bioreactor to reveal or alter metastaticpotential of the tumor cells. In variations of this embodiment, thebioreactor comprises a cell-supporting but cell-permeable matrixseparating at least two fluid chambers with fluid flowing therethroughand at least one gas chamber connected to each of the fluid chambers andsaid tumor cells have been introduced into one of the at least two fluidchambers containing suitable nutrient medium and gas sufficient tosustain tumor growth. In further variations of this embodiment,manipulating the culture comprises altering of one or more of suitablenutrient concentration, oxygen concentration and acidity or comprisesadministering one or more of test compounds, antibodies or biologics.

In yet another embodiment, the invention is a method of identifying anagent capable of inhibiting or stimulating tumor metastasis comprising:preparing a suspension of cells derived from a tumor; introducing asample of the suspension into a first fluid chamber of a bioreactor, thebioreactor, comprising a cell-supporting but cell-permeable matrixseparating at least two fluid chambers with fluid flowing therethroughand at least one gas chamber connected to each of the fluid chambers;supplying to said at least two fluid chambers fluid culture media andgas composition suitable to support tumor growth; incubating thebioreactor under conditions and for a time sufficient for cellproliferation and formation of metastatic cells; introducing a candidateagent into the first or the second chamber; collecting cells that havemigrated into the second chamber; identifying and monitoring thefraction of metastatic tumor cells among the collected cells. Invariations of this embodiment, the agent is selected from among asmall-molecule compound, an antibody or a biologic.

In yet another embodiment, the invention is a method of assessingmetastatic potential of a tumor comprising: preparing a suspension ofcells derived from the tumor; introducing a sample of the suspensioninto a first fluid chamber of a bioreactor, the bioreactor, comprising acell-supporting but cell-permeable matrix separating at least two fluidchambers with fluid flowing therethrough and at least one gas chamberconnected to each of the fluid chambers; supplying fluid culture mediaand gas composition supportive of tumor growth to said at least twofluid chambers; incubating the bioreactor under conditions and for atime sufficient for cell proliferation and production of metastaticcells; collecting cells that have migrated into the second chamber andidentifying metastatic tumor cells among the collected cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a microscopic image of cell culture produced in Example 1.

FIG. 2 shows glucose consumption by the cell cultures produced inExample 2.

FIG. 3 shows lactic acid production by the cell cultures produced inExample 2.

FIG. 4 shows the rate of cell migration out of the cultures produced inExample 2.

FIG. 5 shows CTC and CTPC production (including as proportion of viablecells) in the cultures of Example 2.

FIG. 6 shows glucose consumption by the cell cultures produced inExample 3.

FIG. 7 shows lactic acid production by the cell cultures produced inExample 3.

FIG. 8 shows CTC production by the cultures in Example 3.

FIG. 9 shows CTPC production by the cultures in Example 3.

FIG. 10 shows relative CTC production by the cultures in Example 3.

FIG. 11 shows relative CTPC production by the cultures in Example 3.

FIG. 12 shows glucose consumption by the cell cultures produced inExample 4.

FIG. 13 shows lactic acid production by the cell cultures produced inExample 4.

FIG. 14 shows the rate of cell migration out of the cultures produced inExample 4.

FIG. 15 shows the relative rate of CTC and CTPC production by thecultures in Example 4.

FIG. 16 shows metabolic profile of Capan-2 cell line grown in Example 5.

FIG. 17 shows metabolic profile of MIA PaCa-2 cell line grown in Example5.

FIG. 18 shows the rate of cell migration out of the cultures produced inExample 5.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate the understanding of this disclosure, the followingdefinitions of the terms used herein are provided.

The term “RealBio D4™ Culture System” is a trade name of a bioreactormarketed by RealBio® Technology, Inc. (Kalamazoo, Mich.).

The term “bioreactor” refers to a device that supports a biologicallyactive environment wherein cells or tissues can be grown ex vivo.

The term “cancer cells” and “tumor cells” are used interchangeably torefer to cells derived from a cancer or a tumor, or from a tumor cellline or a tumor cell culture.

The term “metastatic cells” or “metastatic tumor cells” refers to thecells that have the ability to produce a metastasis.

The term “stem cells” refers to multi-potent or pluripotent cellscapable of getting rise to many other cell types.

The term “progenitor cells” refers to undifferentiated cells destined toproduce a specific cell type.

The term “circulating tumor cells” or “CTCs” refers to tumor cells foundin circulation of a patient having a tumor. This term typically does notinclude hematological tumors where the majority of the tumor is found incirculation.

The term “circulating tumor progenitor cells” or “CTPCs” refers to tumorcells found in circulation of a patient having a tumor that are not yetfully differentiated to the point of expressing all characteristics ofmature tumor cells.

The term “cancer stem cells” refers to cells found within tumors thatpossess characteristics associated with normal stem cells includingtheir ability to give rise to all cell types found in a particular tumorsample.

The term “matrix” or “scaffold” are used interchangeably to refer tosolid material that provides support for cells and tissues growing in abioreactor.

The term “primary tumor” refers to a tumor growing at the site of thecancer origin.

The term “metastatic tumor” refers to a secondary tumor growing at thesite different from the site of the cancer origin.

The term “migration” means observable displacement of cells in athree-dimensional space. As used herein, “migration” means both activemigration as well as passive migration of cells.

The term “cell line” refers to a population of cells that through cellculture, has acquired the ability to proliferate indefinitely in vitro.

The term “primary cell culture” refers to a cell culture establishedfrom an organism in the course of a study. A primary cell culture may ormay not give rise to a cell line.

The term “established cell line” refers to a cell line propagated invitro multiple times prior to a study.

The term “metabolic parameter” refers to a parameter reflective of themetabolism of the cells in a culture.

The term “biomarker” refers to a biological marker characterizing aphenotype. A biomarker typically includes a gene or a gene product.Depending on the gene, “detecting a biomarker” may include detectingaltered gene expression, epigenetic modifications, germ-line or somaticmutations, etc. In case of a gene product, “detecting a biomarker” maymean detecting the presence, quantity or change in quantity of a cellsurface marker, a soluble compound such as cytokine, etc. “Detecting abiomarker” may also include detecting a metabolite reflective of agene's expression or activity.

The term “tumor biomarker” or “cancer biomarker” refers to a biomarkercharacteristic of a tumor or cancer but not normal tissue.

The term “small molecule” or “small-molecule compound” refers to a lowmolecular weight non-polymeric organic compound that has (or is beingtested for having) beneficial pharmacological and therapeutic propertiestypically including binding with high affinity to a biopolymer such asprotein, nucleic acid or a polysaccharide and altering the activity orfunction of the biopolymer. The upper molecular weight limit for a smallmolecule is approximately 800 Daltons.

The term “biologic” refers to a biologic medical product that has (or isbeing tested for having) beneficial pharmacological and therapeuticproperties that has been created by a biological process rather thanchemically synthesized. Biologics include for example, blood components,living cells and recombinant proteins.

The invention provides a bioreactor with mixed populations of cancercells (i.e., tumor culture) under appropriate system parameters forgrowing tumor tissues in a three-dimensional arrangement replicating thetumor state in vivo. More particularly, the mixed cancer cellpopulations are grown in such a manner that they maintain the metastaticpotential existing in vivo so that small changes in the systemparameters can stimulate or suppress release of metastatic cells.Release of these cells may be stimulated or suppressed by exposing themixed cancer cell cultures to metastasis triggers such as hypoxia,nutrient deprivation, changes in acidity and other biological orchemical stimulants, or by exposing the mixed cancer cell cultures tometastasis inhibitors. In the course of the culture growth, cellsmigrating out of the culture into the circulating medium may becollected, counted and analyzed for metastatic potential. Similarly,after a change in system parameters, the migrating cells may becollected and analyzed to measure the effect of the change onmetastasis.

The bioreactor used in the present invention supports continuousproduction and output of tumor cells and possibly, metastatic tumorcells over extended periods of time, up to several months. A suitablebioreactor is typically composed of a matrix or scaffold for cellattachment or immobilization; one or more fluid chambers bathing thecell scaffold from above and below while allowing metabolic gases todiffuse; and one or more gas chambers for supplying gases to the fluidchambers.

In some embodiments, the bioreactor comprises two fluid chambersseparated by a matrix for receiving cells, wherein the cells are seededin one chamber, and each fluid chamber is connected to a gas chamber. Insome embodiments, the first and second fluid chambers of the bioreactorare configured to flow the first and second fluids respectivelytangentially to the surface of the matrix material. In some embodiments,the first and second gassing chambers of the bioreactor are operablylinked to the first and second fluid chambers providing gas to the fluidchambers. In some embodiments, each gassing chamber is separated fromthe fluid chamber by a gas permeable membrane positioned between thefluid chamber and the gas chamber.

An example of a suitable device is provided in U.S. Pat. No. 7,682,822,which is hereby incorporated in its entirety, by reference. In someembodiments, the bioreactor is the RealBio D4™ Culture System (RealBioTechnology, Inc., Kalamazoo, Mich.) The RealBio D4™ Culture System is abioreactor designed to recreate a natural, in vivo-like environment forculturing cells. The bioreactor is used to create “ex vivo generatedtissue” or a three-dimensional culture of cells that mimics biologicalproperties of naturally occurring tissue such as for example, normalliver, kidney, gastrointestinal, respiratory, cardiac, adipose, and skintissues as well as tumors derived from these tissues. The bioreactorcombines an open three-dimensional cell scaffold or matrix, perfusednutrient medium, and a mechanism for controlling metabolic gas exchangedecoupled from nutrient delivery. Combined, these features allowresearchers to establish in vivo-like nutrient and gas gradients acrossthe cultured tissues.

In one embodiment, the bioreactor used in the present invention utilizesa three-dimensional matrix to create and maintain a mixed population ofcells simulating a tumor found in a human or other mammalian body.Tumors include without limitation, melanoma, hereditary non-polyposiscolorectal cancer (HNPCC) tumors, nervous system tumors such asneuroblastoma, glioblastoma and retinoblastoma, various carcinomasincluding colon, gastric, pancreatic, renal, ovarian, prostate, breast,cervical, medullary and papillary thyroid carcinoma, non-small cell lungcarcinoma (NSCLC) and adenocarcinoma and various sarcomas includingrhabdomyosarcoma and osteosarcoma. In addition, metastatic tumors thathave developed from various primary tumors are also included in thescope of the present invention.

The matrix can be manufactured from an inert material such aspolystyrenes, polycarbonates or polyesters, including biodegradablepolyesters such as, e.g., polycaprolactone. Other examples of suitablematrix materials include plastic, glass, ceramic or natural biomatrixmaterials such as collagen, alginates, proteoglycans and laminin. Thethree-dimensional matrix may be manufactured from one or both ofnon-woven and woven fibers, having an ordered or random fiberarrangement. An example of a suitable non-woven fabric having a randomfiber arrangement is polyester material such as a felt fabric formedfrom polyethylene terephthalate (PET). In some embodiments, the matrixmember is a three dimensional matrix manufactured from a polyesterfiber, which has a random fiber arrangement. In some embodiments, thematrix may have pores of any size suitable to permit thethree-dimensional growth while also permitting cells to migrate throughthe matrix.

Thickness and density of matrix fibers and the size of pores optimal foreach tumor and cell type may be selected empirically. In certainembodiments, the thickness of a matrix member ranges from about 0.1 toabout 3 mm. The matrix may have pores ranging in size from about 10 toabout 300 microns.

In some embodiments, the invention comprises the use of a bioreactor toestablish a three-dimensional tumor culture that has retained itsnatural ability to metastasize, i.e. shed metastatic cells (includingCTC and CTPC). The tumor culture is established from tumor cells. Inthis embodiment, the tumor cells may be obtained from primary ormetastatic tumors obtained directly from patients or as commerciallyavailable xenografts. In addition, tumor cells may be obtained fromprimary tumor cultures or established tumor cell lines. Solid tumors maybe processed by either mechanical or complete or partial enzymatic orchemical dissociation or a combination of these techniques until asuspension of single cells or multi-cell tissue fragments of desiredsize is obtained. Enzymatic digestion may be carried out by acombination of one or more proteases and nucleases known in the art.After processing, the seeding suspension of cells or multi-cell tissuefragments is introduced into the bioreactor. One or more cells seededinto the bioreactor may represent one or more cell types present in atumor.

In variations of this embodiment, the bioreactor may be prepared toreceive the seeding suspension. For example, the bioreactor may beequilibrated by perfusion with nutrient medium and gases. In someembodiments, the bioreactor is equilibrated to typical conditions forculturing human cells: 37° C. and 5% CO₂. In some embodiments, thematrix may be pre-treated with cell attachment factors such as collagenor laminin. After equilibration, the bioreactor may be seeded with theseeding suspension. In some embodiments, the suspension is introducedinto RealBio D4™ Culture System.

In some embodiments, the invention comprises the use of a bioreactor tomaintain and propagate a three-dimensional tumor culture while the tumorcontinues to metastasize, i.e. shed metastatic progenitor cells(including CTCs and CTPCs). In variations of this embodiment, after thebioreactor is seeded with a seeding suspension, the bioreactor may beretained in desired orientation optionally without perfusion to allowcells to settle into the culture scaffold. After the settling period,the bioreactor may be repositioned in a different orientation. In someembodiments, the bioreactor may be placed on an incline to facilitateseparation of non-adherent cells by gravity. After the settling period,medium flow may also be initiated. In some embodiments, the bioreactormay be placed on an incline and pulsed medium flow may be initiated. Insome embodiments, the cultures are maintained in the RealBio D4™ CultureSystem placed on a 45° incline with a pulsed medium flow cycle.

Cultures may be monitored to confirm growth and tumor expansion. Asdescribed in greater detail below, the growth may be monitored e.g. bymeasuring the increase in the rate of nutrient utilization or wasteproduction. In some embodiments, the growth is monitored by measuringthe rate of glucose consumption or lactate production. In furtherembodiments, the growth may be monitored by measuring concentration ofadditional metabolites including e.g., glutamine, urea, bicarbonate,ammonia, amino acids, lipids, proteins and sugars. The growth may alsobe monitored by withdrawing samples of tumors to determine viable cellcount by any of the techniques known in the art. The cultures may becontinued for several days, weeks or months.

The invention allows for generation of enriched populations ofmetastatic cells for subsequent study. Tumors in vivo generate andrelease metastatic cells (including CTCs and CTPCs). In circulation,metastatic cells become diluted by the large volume of blood and bodyfluids such as lymph. These cells are very rare compared to normal cellsin circulation. In contrast, the bioreactor has a much smaller volumefrom which the sloughed cells are collected. Furthermore, the bioreactordoes not contain additional cell types, e.g. white and red blood cellsnormally present in circulation alongside with metastatic cells. Thus inthe bioreactor, the concentration of released metastatic cells is muchhigher and they can be collected much easier from relatively small tumorspecimens without the need for high efficiency cell separationtechnologies.

In some embodiments, the sloughed cell population comprising metastaticcells may be continually or periodically removed from the bioreactor.The cells may be removed via a harvest port engineered into a fluidchamber of the bioreactor.

In some embodiments, the invention comprises the use of a bioreactor toharvest and analyze metastatic cells including circulating tumor cells(CTCs) and circulating tumor progenitor cells (CTPCs). In thisembodiment, samples of sloughed cells are taken at different stages maybe analyzed and compared. In some embodiments, a sample of whole bloodfrom the animal or patient bearing the tumor used to initiate thecultures, a sample of the dissociated tumor suspension used to seed theculture systems, and the samples collected from the bioreactor may beanalyzed and compared. Furthermore, upon termination of the culture, thematrix or scaffold may be excised from the bioreactor for examination oftissue development by direct staining, traditional histologicalprocessing and scanning electron microscopy (SEM). For microscopicanalysis the sample may be stained for example, with hematoxylin andeosin (H&E) or other differential stains, e.g., PROTOCOL® HEMA 3staining. All cells may be stained with the fluorescent nucleicacid-binding dye, such as Hoechst 33342 or DAPI to aid indifferentiating cells from cellular debris. Cells exhibiting positivestaining with the various markers described below may be identified asCTCs or CTPCs, counted and further characterized.

Several biomarkers as well as morphological, immunological andphysiological tests or combinations thereof may be used to identify CTCsand CTPCs. See e.g. Sun et al. (2011), Circulating tumor cells: advancesin detection methods, biological issues, and clinical relevance, J.Cancer Res. Clin. Oncol. 137:1151-1173; Man, et al. (2011), Currentlyused markers for CTC isolation—advantages, limitations and impact oncancer prognosis, J. Clin. Exper. Pathol. 1:1. For example, CTCs andCTPCs may be identified by their ability to adhere to cell adhesionmolecules (CAM), as well as by the presence of certain specificbiomarkers including EpCAM, cytokeratins (CK) 5, 7, 18 and 19. Dependingon the tumor of origin, CTCs may also be identified based on thepresence of tumor-specific biomarkers including IGF-1R, BCL2, HER2,EphB4, CA19-9, CEA, CD133, MUC1, Survivin and PTEN. For example, CTCsoriginating from the pancreas would exhibit positive staining withstandard epithelial markers and human pancreatic tumor markers (EpCAMand CA19-9). CTPCs may be identified in a similar fashion except thattumor progenitor markers (CD44v6, N-cadherin, and FAP (Seprase)) may beused in the place of epithelial markers. In some embodiments of theinvention, CTCs and CTPCs are identified using VITA-ASSAY™ AR16 platform(Vitatex, Inc., Stony Brook, N.Y.).

In some embodiments, the invention comprises the use of a bioreactor fortesting the collected cells for their capacity to form metastaticlesions in healthy tissues. This embodiment may further comprisestudying the processes related to the development of metastases. In thisembodiment, collected cells may be infused into additional bioreactorsin which cultures of mixed cell populations representing healthy“target” tissues have been established. In some embodiments, the studyof the metastatic process comprises characterizing cells collectedfollowing changes in system parameters (e.g. changes in oxygenconcentration, pH, nutrients etc.) by genetic analysis (e.g. for thepresence of biomarkers described above), in vitro invasion assays, cellmarker-based tumor progenitor identification assays, anti-cancer drugresponse assays, as well as other established methods for identifyingand characterizing metastatic cells. Samples of the circulating mediummay also be analyzed directly for changes in soluble metastasis-relatedbiomarkers in response to changes in system parameters.

The invention allows the effects of single or combinations of cultureparameters to be studied. For example, the nutrient and oxygen levels inthe system may be dropped in concert to simulate conditions that arethought to stimulate metastatic behavior in large tumors. In someembodiments, ports are integrated into one or more of the fluid chamberinputs to deliver liquid components to the bioreactor. In addition tonutrients, anti-metastasis agents, immunological factors and anti-cancercompounds and biologics, including compounds that modulate geneexpression and cell function such as cytokines, toxins, nucleic acids(e.g., microRNA), or other cell types may also be added to the perfusionfluids of the bioreactor to study the effect of treatment with thesecompounds as single agents or combinations.

In some embodiments, the invention comprises use of a bioreactor tocreate an in vitro model of a patient's cancer wherein the cancer hasretained its metastatic potential which can aid in the selection ofpersonalized anti-cancer treatments that would prevent or eliminatemetastases in the patient.

In some embodiments, the invention comprises the use of a bioreactor todetermine physiological characteristics of a tumor culture in relationto the tumor's ability to metastasize. In this embodiment, therecoverable suspension of the tumor culture maintained in the bioreactormay be periodically sampled and analyzed. In other variations of thisembodiment, a sample may be retrieved from a compartment separated fromthe tumor culture for example, by the matrix separating the chambers ofthe bioreactor, such as the bottom chamber. In some embodiments, thesample is withdrawn from the bottom compartment of the RealBio D4™Culture System. The analysis of culture parameters (including glucoseand lactate concentration, migrating cell numbers) may be performed atthis time. Glucose concentration may be measured using any technique anddevice known the art, for example using ACCU-CHEK® Aviva blood glucosemonitor (Roche Diagnostics Corp., Indianapolis, Ind.). Lactate similarlymay be measured using any technique and device known the art, forexample using the Lactate Plus test meter (Nova Biomedical Corp.,Waltham, Mass.). The total cell density of each sample may also beestimated using any technique and device known the art, for exampleusing the TC10™ Automated Cell Counter (Bio-Rad Labs., Hercules, Cal.).

According to the present invention, tumors can be cultured in thebioreactor in any physiologically acceptable liquid culture medium.Guidance for selecting culture medium and conditions may be found inSandell, L. and Sakai, D. Mammalian Cell Culture. Current ProtocolsEssential Laboratory Techniques 5:4.3.1-4.3.32, John Wiley & Sons, 2011.A medium optimal for a particular tumor type may be empirically foundamong the many commercially available products including AIM V, IMDM,MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's Medium. The medium can besupplemented with serum as known in the art, typically at 1% to 50%.Alternatively serum substitutes comprising serum albumin, cholesterol,lecithin and inorganic salts may be used. The tumor cultures aretypically carried out at a pH which approximates physiologicalconditions, between 6.9 and 7.4. The medium is typically exposed to anoxygen-containing atmosphere which contains from 2 to 20% oxygen. Insome embodiments, the parameters are altered to simulate hypoxia,acidosis, nutrient starvation, accumulation of waste products and otherpathological conditions known to occur in tumors.

The invention further includes a method and system for selecting andtesting anti-tumor and anti-metastasis agents including compounds,antibodies and biologics. Such candidate agents may be introduced intothe bioreactor cultures of the present invention and tested for theirability to alter the production of metastatic cells (including CTCs andCTPCs) by the cultures.

The invention provides the flexibility to present different nutrient andgas conditions on each side of the cultured tumor tissue in thebioreactor. In some embodiments, independently, oxygen-rich andoxygen-poor gases may be supplied to the two sides of the culturesystem. Since manipulation of such nutrient and gas gradients isnecessary for gaining better understanding of the role that the tumormicroenvironment plays in tumor development, growth and metastasis; andsince these gradients cannot be readily established and manipulatedusing other in vitro technologies or within in vivo studies; thisfeature of the invention provides a significant advantage over existingtechnologies.

EXAMPLES Example 1

Establishment of a Metastatic Pancreatic Tumor In Vitro

A metastatic pancreatic tumor (pancreas to liver) weighing approximately1 gram was minced and partially dissociated enzymatically beforeapproximately ⅕ of the dissociated mass was infused into a RealBio D4™Culture System bioreactor. The mixed population of tumor and associatedstromal cells was maintained in the bioreactor by circulating Iscove'sModified Dulbecco's Medium (IMDM) supplemented with fetal bovine serum(FBS) (10%) and antibiotics through both the upper and lower fluidchamber. The bioreactor was maintained in an incubator at 37° C. with a5% CO₂ environment. Samples of the culture medium were collected fromthe lower compartment of the bioreactor three times per week to countthe number of cells shed by the cultured tumor and to monitor metabolicactivity of the culture (glucose consumption and lactate production).After 29 days in culture, a sample of the cells migrating out of thecultured tumor was analyzed to identify and enumerate circulating tumorcells (CTCs) and circulating tumor progenitor cells (CTPCs) usingVITA-ASSAY™ AR16 platform (Vitatex, Inc., Stony Brook, N.Y.). Scanningelectron microscopy (SEM) was used to examine the cultured tumors fromwhich the CTCs and CTPCs have migrated.

The results are shown on Table 1 and FIG. 1. The sample which included amixture of live and dead cells of tumor and stromal derivation wassignificantly enriched for CTCs and CTPCs (Table 1) relative to theextremely low frequency of CTCs and CTPCs typically observed in blood invivo, despite the absence of any treatments aimed at inducing oraugmenting release of metastatic cells.

TABLE 1 Relative Abundance of Circulating Pancreatic Cancer Tumor CellsSample CTCs CTPCs Typical blood from patient w/metastatic cancer <0.001%<0.001% (Data from National Pancreas Foundation) Metastatic tumor in theRealBio D4 ™ Culture 2% 3% System Relative abundance expressed aspercentage of total cells or cell-like particles

Electron microscopy of the tumor culture 28 days after cells were seededinto the culture chamber (FIG. 1) confirms the persistence of multiplecell types (shown by arrows).

Example 2 In Vitro Production of Circulating Tumor Cells (CTCs) byMetastatic Pancreatic Tumor Tissue

Metastatic pancreatic tumor tissue was obtained as a fresh mousexenograft tumor (P1) from a commercial source. The xenograft tumor wasoriginally derived from a human adenosquamous carcinoma of the pancreasthat had metastasized to the liver of a 46 year old female. The tumorwas excised from the host animal and shipped overnight on “blue ice”cold packs in serum-free RPMI culture medium containing penicillin andstreptomycin. Upon receipt, the entire tumor mass (wet weight=1.21 g)was immediately processed by mechanical and partial enzymaticdissociation using LIBERASE™ and DNase I (Roche Applied Science,Indianapolis, Ind.)

RealBio D4™ Culture System bioreactors configured with a single,recirculating flow loop were primed with 35 mL of complete culturemedium and equilibrated overnight in a standard CO₂ incubator (passivegassing) at 37° C., 5% CO₂. A total of six bioreactors representingduplicates of three minor culture chamber variations were used (Table2). The minor variations between the test groups involved differentorientations of a single, woven synthetic scaffold material with orwithout surface plasma treatment of the scaffold fibers to evaluate theeffect of different scaffold conditions on culture establishment.

TABLE 2 RealBio D4 ™ Culture System Bioreactor Configurations Test GroupScaffold Type Scaffold Comments 1 A Standard orientation, untreated 2 AInverted, untreated 3 B Standard orientation, plasma treated

For seeding, medium flow was suspended and bioreactors were placed in ahorizontal orientation. 3 mL of the dissociated tumor suspension wasinfused into the top compartment of each bioreactor. After a 24 hrsettling time with no medium flow, the bioreactors were placed on a 45°incline and pulsed medium flow was reinitiated. Samples were collectedfrom the bottom compartment of each bioreactor 3 times per week.Approximately 100 μL of each sample was separated for analyzing routineculture parameters (glucose and lactate concentration, migrating cellnumbers) while the remainder of each sample was reserved for CTC andCTPC analysis as described below.

One culture from each test group was terminated after 19 days and theremaining culture after 42 days. Upon termination, the fabric scaffoldwas excised from each bioreactor and divided into sections forexamination of tissue development by direct staining, traditionalhistological processing and scanning electron microscopy (SEM) asdescribed below.

Glucose concentration was measured using the ACCU-CHEK® Aviva bloodglucose monitor (Roche Diagnostics Corp., Indianapolis, Ind.) andlactate concentration was measured using the Lactate Plus test meter(Nova Biomedical Corp., Waltham, Mass.). The total cell density of eachsample was estimated using the TC10™ Automated Cell Counter (Bio-RadLabs., Hercules, Cal.) without trypan blue staining.

Identification and enumeration of Circulating Tumor Cells (CTCs) andCirculating Tumor Progenitor Cells (CTPCs) was performed using theVITA-ASSAY™ AR6W platform. The samples analyzed included a sample ofwhole blood from the mouse bearing the tumor used to initiate thecultures, a sample of the dissociated tumor suspension used to seed theculture systems, and the samples collected periodically from the lowercompartment of the culture chambers. For all samples, VITA-ASSAY™identifies viable CTCs using EpCAM and CA19-9. Viable CTPCs wereidentified in a similar fashion except that tumor progenitor markers(CD44v6 and FAP (Seprase)) were used in the place of epithelial markers.All cells were also stained with the fluorescent nucleic acid dyeHoechst 33342 to aid in differentiating cells from cellular debris.Cells exhibiting positive staining with the various markers were countedmanually under multi-parametric fluorescence microscopy. Sections of thefabric scaffold excised from cultures after termination on days 19 and42 were processed by staining directly (HEMA 3 stain), or staining withH&E (after paraffin embedding and sectioning), or by SEM.

The progress of the cultures was assessed by glucose and lactateanalyses shown in FIGS. 2-3. FIG. 2 illustrates the rate of glucoseconsumption between sampling intervals for each test group defined inTable 2. Values for intervals through Day 19 represent the average fromduplicate cultures while values from intervals beyond Day 19 are fromsingle cultures only. Glucose consumption rates increased dramaticallyand to a similar extent across the entire duration of the study forcultures from all three test groups suggesting that scaffold type andorientation had minimal impact on the rate of culture establishment andexpansion (FIG. 2). FIG. 3 illustrates the concentration of lactate inthe circulating culture medium for each test group defined in Table 2.Values for time points through Day 19 represent the average fromduplicate cultures while values from time points beyond Day 19 are froma single culture only. The vertical drops on days 7, 13, 17, 21, 23, 28,33 and 40 represent dilution of lactate due to culture feeding (partialmedium exchanges). Likewise, no difference was seen between test groupswith respect to lactate production (FIG. 3).

Microscopic examination revealed that tumor cultures were successfullyestablished in all six of the RealBio D4™ Culture Systems seeded withdissociated tumor tissue regardless of the variation of scaffoldmaterial or orientation. Observations on day 19 revealed an evendistribution of cells across the top of scaffolds in Test Groups 1 and 2with less than full scaffold coverage. The distribution of cells acrossthe top of the scaffold from Test Group 3 was uneven, with some denselycovered areas and some areas essentially devoid of cells. The undersideof the scaffolds from all test groups was sparsely populated on Day 19.By Day 42, the top and bottom surfaces of scaffolds from all test groupswere completely covered with cells. HEMA 3 staining revealed multiplecell types in all cultures.

The total number of cells migrating from the cultures in each test groupwas evaluated 3× per week and normalized with respect to the number ofdays between sampling (FIG. 4). For FIG. 4, the number of cellsharvested from the lower compartment of each bioreactor was normalizedon a per day basis for each test group. Values for time points throughDay 19 represent the average from duplicate cultures while values fromtime points beyond Day 19 are from a single culture only. Gaps in eachprofile represent cell numbers below the threshold the cell countinginstrument. After the first week in culture, the migration ratesstabilized at fewer than 2×10⁵ cells/day for each test group.

For comparison, the analysis of CTCs and CTPCs in mouse tissues (wholeblood, 0.8 mL, and the dissociated tumor suspension used to seed thebioreactors, 0.5 mL) is shown in Table 3. The number of CTCs and CTPCscollected from the lower compartment of tumor cultures across theduration of the study is detailed in Table 4. The rate of CTC and CTPCproduction is shown in FIG. 5. On FIG. 5, the mean sum of CTC and CTPCcell counts (excluding clusters) across the three test groups is shownafter being normalized to account for the number of cultures sampled ateach time point and the number of days between sample collections, andas a percentage of the total viable cell count. On each plot, the firstfour data points represent samples that were frozen prior to analysisand exhibited poor viability, while the last three data points representsamples processed without freezing resulting in significantly higherviability.

TABLE 3 CTC

 CTPC Analysis of Mouse Tissue Samples CTC CTPC Sample CTCs ClustersCTPC Clusters Blood (0.8 mL) 30 0 16 2 Dissociated 20 0 16 0 Tumor (0.5mL)

Fresh blood sample (not frozen) was analyzed ˜48 hours after collection.<1% of tumor cells were viable upon thawing prior to analysis.

TABLE 4 CTC 

 CTPC Analysis of Cells Migrating from Tumor Cultures Cell Count CTCCTPC Test (Viable/ Clus- Clus- Group Sample Total) CTCs ters CTPCs ters1 Day 2, 5, 7 <1%/nd    70 4 44 0 Pool^(1,2) Day 9, 13, <1%/nd    44 220 0 15 Pool^(1,2) Day 17, 19, 1360/12,600 0 0 4 2 21 Pool^(1,2) Day 23,26, 1340/10,800 26 2 30 4 28 Pool¹ Day 33 2,230/2,800  32 0 40 0 Day 351,700/8,500  54 0 46 0 Day 40 190/1,000 74 0 44 0 2 Day 2, 5, 7<1%/nd    166 0 76 2 Pool^(1,2) Day 9, 13, <1%/nd    18 0 16 0 15Pool^(1,2) Day 17, 19, 880/2,600 6 0 10 0 21 Pool^(1,2) Day 23, 26,740/3,000 0 0 28 4 28 Pool¹ Day 33 2,450/2,800  52 0 48 0 Day 351,420/3,500  62 0 50 0 Day 40 740/2,000 84 2 64 4 3 Day 2, 5, 7<1%/nd    80 0 74 0 Pool^(1,2) Day 9, 13, <1%/nd    12 0 6 0 15Pool^(1,2) Day 17, 19,  340/15,600 6 2 20 2 21 Pool^(1,2) Day 23, 26, 640/10,200 32 2 8 0 28 Pool¹ Day 33 3,350/3,500  140 0 92 0 Day 35980/2,000 46 0 34 0 Day 40 280/1,500 160 0 122 0 ¹Individual sampleswere frozen and thawed prior to pooling. ²Samples were collected fromduplicate cultures through Day 19 but single cultures thereafter.

Example 3

Effect of Hypoxia on In Vitro Production of Circulating Tumor Cells(CTCs) by Metastatic Pancreatic Tumor Tissue

Metastatic pancreatic tumor tissue was obtained as a fresh mousexenograft tumor (P1) from a commercial source. The xenograft tumor wasoriginally derived from a stage IV metastatic adenocarcinoma of thepancreas that had metastasized to the peritoneum of a 78 year old malepatient. Upon receipt, the tumor was processed by mechanical and partialenzymatic dissociation and the RealBio D4™ Culture System bioreactorswere seeded essentially as described in Example 2. Duplicate bioreactorswere prepared for each of four test groups differing only with respectto the concentration and mode of oxygen delivery (Table 5).

TABLE 5 RealBio D4 ™ Culture System Bioreactor Configurations Test GroupCell Type Gas Supply 1 Primary Passive, Normoxia Tumor 2 Primary Active,Normoxia Tumor 3 Primary Active, Moderate Hypoxia Tumor 4 PrimaryActive, Normoxia (first 20 Days) Tumor Active, Moderate Hypoxia (last 31Days)

Passive delivery of oxygen at the ambient concentration (˜21%) wasaccomplished by placing culture chambers in a standard, humidified CO₂incubator at 37° C. with 5% CO₂. Active delivery of oxygen at either 2or 20% was accomplished by perfusing humidified premixed gas (2% O₂/5%CO₂/93% N₂ or 20% O₂/5% CO₂/75% N₂). No difference was observed in thedissolved oxygen concentrations measured in culture systems configuredwith active gassing using 20% oxygen or passive ambient gassing(measured dissolved oxygen concentration≈21% in each case, i.e.,normoxia). The concentration of dissolved oxygen was determined using anISO₂ dissolved oxygen meter (World Precision Instruments, Inc.,Sarasota, Fla.).

To assess the progress of the cultures, glucose and lactic acidconcentrations were determined and the total cell density of samplescollected from the bioreactor was estimated essentially as described inExample 2. Results are shown in FIGS. 6-7. Glucose consumption ratestrended upwards for all cultures across the duration of the study,although significant fluctuations in glucose consumption rates thatcorrelated with routine feeding intervals were present (FIG. 6). Thesteady state lactate concentration of 18 mM was observed for all tumorcultures (FIG. 7). Note the relatively smooth and consistent profilesobserved for all test groups despite significant lactate dilution as aresult of partial medium exchanges (feeding) on days 3, 10, 17, 24, 27,31, 38 and 45. The cultures from Test Groups 1 and 3 (passive ambientoxygen delivery and moderately hypoxic conditions, respectively)demonstrated consistently higher glucose consumption rates and lactateconcentration between days 10 and 20 compared to the other culturessuggesting that there was a genuine enhancement of metabolic ratesduring this portion of the study. These differences disappeared,however, during the latter half of the study as the glucose consumptionand lactate production rates for Test Groups 2 and 4 (active oxygendelivery and active oxygen delivery followed by hypoxia, respectively)rose to meet those of Groups 1 and 3 in the final 30 days of the study.

One culture from each test group was terminated after 20 days and theremaining culture after 51 days. Upon termination, the fabric scaffoldwas excised from each bioreactor and divided into sections forexamination of tissue development by direct staining and scanningelectron microscopy essentially as described in Example 2.

Microscopic examination revealed that the concentration of oxygen duringthe first 19 days of the study affected the distribution of cells acrossthe culture scaffold. Large, dense “colonies” of cells with sparse cellpopulations in between were observed across the upper surface of thescaffolds from all of the cultures exposed to normoxia (passive oractive delivery) while a more uniform cell distribution was observedacross the scaffold from the culture that was maintained undermoderately hypoxic conditions. The underside of the scaffolds from alltest groups was sparsely populated. These distribution patternspersisted through the end of the study (51 days).

Identification and enumeration of CTCs and CTPCs was performedessentially as described in Example 2. Results are shown in Table 6.

TABLE 6 CTC 

 CTPC Analysis of Cells Migrating from Tumor Cultures Cell Count Test(Viable/ CTC CTPC Group Sample Total) CTCs Clusters CTPC Clusters 1 Day20 2700/7560  92 0 96 0 Passive, Day 20 2100/5400  50 0 34 0 NormoxiaDay 27 230/840  6 0 2 0 Day 34 180/1400 0 0 4 2 Day 41 260/1960 8 0 12 02 Day 20 610/1400 2 0 2 0 Active, Day 20 770/1680 4 0 0 0 Normoxia Day27 140/360  4 0 2 0 Day 34 250/1960 22 0 30 0 Day 41 980/2800 24 0 68 03 Day 20 1700/3640  2 0 6 0 Active, Day 20 510/2800 4 0 0 0 Moderate Day27 150/280  0 0 0 2 Hypoxia Day 34 170/1120 20 0 8 0. Day 41 440/2520 64 6 0 4 Day 20 600/3360 2 0 4 0 Active, Day 20 550/2800 0 0 0 0 Normoxia→ Day 27 80/280 8 0 2 2 Moderate Day 34 120/1400 12 0 4 0 Hypoxia Day 41290/1960 14 0 6 4

The number of cells migrating from the tumor cultures in each test groupwas evaluated 3× per week and normalized with respect to the number ofdays between sampling. The migrating cell numbers fluctuated around4−5×10⁴ cells per day for each of the test groups with no obviouscorrelation with oxygen levels. The numbers of CTCs and CTPCs was alsoevaluated (FIGS. 8-9). The asterisk for Group 1, Day 20 signifies a moreaggressive sampling technique used for that single sample (value offscale). Oxygen had no clear effects on the number of migrating cellsexpressing the CTC and CTPC phenotype, though the samples from TestGroup 2 (active delivery, normoxia) demonstrated the highest number ofCTCs and CTPCs during the last two sampling intervals tested. There wasno difference in the fraction of CTCs, however, the fraction of CTPCswas elevated in Test Group 2 (FIGS. 10-11).

Example 4 Effect of Hypoxia on In Vitro Production of Circulating TumorCells (CTCs) by Pancreatic Cell Lines

Two human pancreatic cancer cell lines reported to be highly metastaticin mouse xenograft models (MIA PaCa-2 and AsPC-1) and two humanpancreatic cancer cell lines that rarely metastasize in mouse xenograftmodels (PL45 and Capan-2) were obtained from the American Type CultureCollection (ATCC). Each cell line was maintained in T-75 flasks at 37°C., 5% CO₂ using the growth medium recommended by the ATCC prior toculturing in RealBio D4™ Culture Systems. The RealBio D4™ Culture Systembioreactors were prepared and seeded essentially as described in Example2. The bioreactors were configured as described in Table 7. Twobioreactors were prepared for culturing each of the four cell lines (8systems total).

TABLE 7 RealBio D4 ™ Culture System Bioreactor Configurations-Cell LineCultures Cell Type Metastatic Potential Growth Medium AsPC-1 High RPMI1640, 10% FBS Capan-2 Low McCoy's 5a Medium, 10% FBS PL45 Low DMEM, 10%FBS MIA PaCa-2 High DMEM, 10% FBS, 2.5% Horse Serum

One culture from each test group was terminated after 13 days, while theremaining culture from each test group was terminated after 39 days.Upon termination, the fabric scaffold was excised from each culturechamber and divided into sections for examination of tissue developmentby direct staining essentially as described in Example 2.

To assess the progress of the cultures, glucose and lactic acidconcentration was determined and the total cell density of cell culturesperiodically collected from the bioreactors was estimated essentially asdescribed in Example 2. A peak glucose consumption rate of approximately2000 mg/day was observed for the MIA PaCa-2 culture after only 2 weeksof incubation. This rate of glucose utilization was nearly four timesthat of the next most active culture (Capan-2) and approximately fiftytimes higher than that observed for the AsPC-1 culture (FIG. 12).Despite the wide disparity in glucose consumption rates for the fourcell lines, the steady state lactate levels varied by less than a factoror two (FIG. 13). The irregularity of the curves for the MIA PaCa-2 cellline in FIGS. 12 and 13 may relate in part to differences in the glucoseconcentration of the medium supplied to the culture at different timesthroughout the study.

Microscopic examination of culture scaffold sections removed from theculture chambers was performed after 13 and 39 days. MIA PaCa-2 cellsexpanded rapidly in the culture chambers, covering the upper surface ofthe scaffold fabric with multiple cell layers and occluding most of thelarge voids between scaffold fiber bundles by Day 13, though theunderside of the scaffold remained largely unpopulated. By Day 39, veryheavy accumulations of the cells were found on top of the scaffoldmaterial along with moderate to heavy cell densities on the underside.Essentially all of the cells exhibited a rounded morphology whether theyare found attached directly to scaffold fibers or associated with othercells in dense, tissue-like clusters, and although most of the cellswere adherent, a large number of cells could be seen sloughing off theculture scaffold when the medium was drained from the culture chamberfor histological examination on Day 39.

AsPC-1 cultures exhibited moderate cell densities and significantamounts of natural extracellular matrix material across the top of thescaffold fabric after 13 days in culture, and though cell densitiesincreased only modestly after Day 13, many of the larger voids betweenthe fiber bundles of the scaffold fabric were filled with cells by thetime that cultures were terminated on Day 39. The underside of theculture scaffold, however, remained essentially devoid of cells for theduration of the study. The size and shape of AsPC-1 cells appeared moreheterogeneous compared to the relatively uniform morphology of MIAPaCa-2 cells.

Capan-2 and PL45 cells arranged themselves in very similar fashion onthe scaffold material with all cells found very closely associated withscaffold fibers and very few cells spanning open areas between fibers.After 13 days in culture, both of these cell types covered a majority ofthe scaffold fiber bundles on the upper side of the scaffold but thelarge “pores” between fiber bundles remained open. The density of cellson the fiber bundles was higher after 39 days but the vast majority ofscaffold “pores” still remained open and only spotty “ribbons” of cellsclosely associated with scaffold fibers were observed on the undersideof the scaffold. It appeared that the Capan-2 culture exhibited slightlyhigher cell densities overall when compared to the PL45 cell line.Interestingly, no isolated single cells could be found in the Capan-2culture (only very few were observed in the PL45 cultures) and neitherthe Capan-2 nor the PL45 cells produced visible amounts of naturalextracellular matrix material.

Identification and enumeration of CTCs and CTPCs was performedessentially as described in Example 2. Results are shown in Table 8.

TABLE 8 CTC 

 CTPC Analysis of Cells Migrating from Pancreatic Cell Lines Maintainedin the RealBio D4^( ™) Culture System Bioreactors Cell Count Cell(Viable/ CTC CTPC Line Sample Total) CTCs Clusters CTPC Clusters AsPC-1Day 25 7200/25000 1010 0 1080 4 Day 32 5000/8400  306 0 404 0 Capan-2Day 25 8600/56000 16 0 22 0 Day 32 13600/28000  24 0 22 0 PL45 Day 251640/45000 50 0 16 0 Day 32 880/4200 16 0 10 0 MIA Day 25 80000/36500018196 0 16000 0 PaCa-2 Day 32 6000/30000 1040 0 1260 0

FIG. 14 illustrates the number of cells harvested from the lowercompartment of each bioreactor and normalized on a per day basis foreach pancreatic cancer cell line. Gaps in each profile represent cellnumbers below the threshold of the cell counting instrument. The numberof cells migrating out of the cell line cultures into the bottomcompartment of the bioreactor chambers did not appear significantlydifferent for any of the cell lines until a burst of cells was producedby the MIA PaCa-2 cell line beginning after 25 days in culture. Therelative rate of CTC and CTPC production is shown on FIG. 15.

Example 5

Comparison of in vitro CTC Production by Metastatic and Non-metastaticCell Lines Grown in the RealBio D4™ Culture System Bioreactor underNormoxia and Hypoxia

Two human pancreatic cancer cell lines described in Example 4: highlymetastatic MIA PaCa-2 and rarely metastatic Capan-2 were used. Fourbioreactors were prepared for culturing each cell line (8 bioreactorstotal). The bioreactors were prepared and seeded essentially asdescribed in Example 2. The configuration of bioreactors is shown inTable 9.

TABLE 9 RealBio D4 ™ Culture System Bioreactor Configurations Test GroupCell Type Metastatic Potential Gassing Condition 1 Capan-2 Low Normoxia2 Capan-2 Low Hypoxia 3 MIA PaCa-2 High Normoxia 4 MIA PaCa-2 HighHypoxia

Passive delivery of oxygen at the ambient concentration (˜21%) wasmaintained for the first 10 days of the study by keeping culturechambers in a standard, humidified CO₂ incubator at 37° C. with 5% CO₂.Beginning on Day 10, delivery of oxygen to the cultures was initiatedand performed essentially as described in Example 3. On Day 13, the flowof medium was changed for all cultures from pulsed flow to constant flowat a slower rate (maintaining the overall medium exchange rate withinthe culture chambers).

To assess the progress of the cultures, glucose and lactic acidconcentration, and oxygen concentration in the medium were determinedand the total cell density was estimated essentially as described inExample 2. Glucose consumption rates trended upwards throughout thecourse of the study for both cell lines (FIGS. 16-17). Neither cell lineexhibited obvious changes in lactate production when switched fromnormoxic to hypoxic conditions. Note the relatively smooth andconsistent lactate profiles observed despite significant lactatedilution as a result of partial medium exchanges (feeding) performed ateach sampling interval beginning on Day 5. FIG. 18 illustrates thenumber of cells harvested from the lower compartment of each bioreactorand normalized on a per day basis for each cell line. Gaps in eachprofile represent cell numbers below the threshold of the cell countinginstrument. The switch to hypoxia caused an apparent decrease in thetotal number of cells migrating out of the cultured cell masses for bothcell lines.

Samples were collected from the bottom compartment of each bioreactorand partial medium exchanges were performed 3 times per week. Oneculture from each test group was terminated after 18 days, and theremaining culture after 31 days. Upon termination, the fabric scaffoldwas excised from each culture chamber and examined for tissuedevelopment by direct staining essentially as described in Example 2.

Identification and enumeration of CTCs and CTPCs was performedessentially as described in Example 2. Results are shown in Table 10.

TABLE 10 CTC

 CTPC Analysis of Cells Migrating from 31-Day Cultures Gassing CTCsCTPCs Cell Type Condition (% of viable cells) (% of viable cells)Capan-2 Normoxia  16 (0.1%) 12 (0.1%) Hypoxia  8 (0.1%) 59 (0.9%) MIAPaCa-2 Normoxia 764 (4.2%) 1520 (8.3%)  Hypoxia 712 (4.0%) 620 (3.5%) 

Microscopic examination revealed that under normoxia (20% oxygen), thedensity and arrangement of Capan-2 cells was uniform across the entireculture scaffold. Under hypoxic conditions, the arrangement of cellsvaried across the length of the culture scaffold. The varied growthpattern gradient observed across the scaffold suggests the presence ofan oxygen gradient across the scaffold and therefore a mixture of cellcells growing under varying levels of hypoxia. For MIA PaCa-2 cells noobvious difference was observed in cell morphology or cell arrangements.For both cell lines, the density was notably lower under hypoxicconditions.

While the invention has been described in detail with reference tospecific examples, it will be apparent to one skilled in the art thatvarious modifications can be made within the scope of this invention.Thus the scope of the invention should not be limited by the examplesdescribed herein, but by the claims presented below.

We claim:
 1. A combination of a bioreactor and one or more tumor cellsin which the system parameters are such that the tumor cells maintaintheir normal metastatic potential.
 2. The combination of claim 1,wherein the bioreactor comprises a cell-supporting but cell-permeablematrix separating at least two fluid chambers with fluid flowingtherethrough and at least one gas chamber connected to each of the fluidchambers.
 3. The combination of claim 1, wherein said one or more tumorcells have been introduced into one of the at least two fluid chamberscontaining suitable nutrient medium and gas sufficient to sustain tumorgrowth.
 4. The combination of claim 1, wherein the normal metastaticpotential comprises the ability to produce metastatic cells.
 5. Thecombination of claim 4, wherein the metastatic cells include cellshaving characteristics of circulating tumor cells and circulating tumorprogenitor cells.
 6. The combination of claim 5, wherein thecharacteristics of circulating tumor cells and circulating tumorprogenitor cells include the presence of one or more of the followingbiomarkers: EpCAM, CK5, CK7, CK18, CK19, Cd44v6, EphB4, FAP (seprase),IGF-1R, BCL2, HER2, CA19-9, CEA, CD133, MUC1, N-cadherin, Survivin andPTEN.
 7. A method of generating metastatic tumor cells in vitrocomprising: (a) introducing one or more tumor cells into a bioreactor;(b) providing to the bioreactor fluid culture media and gas compositionsupportive of growth of the tumor cells; (c) incubating the bioreactorunder conditions and for a time sufficient for the tumor cells toproduce metastatic cells; (d) collecting metastatic tumor cells.
 8. Themethod of claim 7, wherein the bioreactor comprises a cell-supportingbut cell-permeable matrix separating at least two fluid chambers withfluid flowing therethrough and at least one gas chamber connected toeach of the fluid chambers.
 9. The method of claim 7, wherein the tumorcells are introduced in the first fluid chamber and fluid culture mediaand gas composition supportive of tumor growth are supplied to said atleast two fluid chambers.
 10. The method of claim 7, wherein conditionsincluding one or more of glucose concentration, lactic acidconcentration and pH are monitored in the bioreactor during step (c).11. The method of claim 7, wherein metastatic tumor cells are collectedfrom among cells that have migrated into said second chamber.
 12. Themethod of claim 7, further comprising confirming the nature ofmetastatic tumor cells by detecting the presence of one or more of thefollowing biomarkers: EpCAM, CK5, CK7, CK18, CK19, Cd44v6, EphB4, FAP(seprase), IGF-1R, BCL2, HER2, CA19-9, CEA, CD133, MUC1, N-cadherin,Survivin and PTEN
 13. A method of manipulating a culture of tumor cellsin a bioreactor to reveal or alter metastatic potential of the tumorcells.
 14. The method of claim 13, wherein the bioreactor comprises acell-supporting but cell-permeable matrix separating at least two fluidchambers with fluid flowing therethrough and at least one gas chamberconnected to each of the fluid chambers.
 15. The method of claim 13,wherein said tumor cells have been introduced into one of the at leasttwo fluid chambers containing suitable nutrient medium and gassufficient to sustain tumor growth.
 16. The method of claim 13, whereinmanipulating the culture comprises altering of one or more of suitablenutrient concentration, oxygen concentration and acidity.
 17. The methodof claim 13, wherein manipulating the culture comprises administeringone or more of test compounds, antibodies or biologics.
 18. The methodof claim 13, wherein the metastatic potential is measured by the numbersof cells produced in the bioreactor that possess one or more of thefollowing biomarkers: EpCAM, CK5, CK7, CK18, CK19, Cd44v6, EphB4, FAP(seprase), IGF-1R, BCL2, HER2, CA19-9, CEA, CD133, MUC1, N-cadherin,Survivin and PTEN.
 19. A method of identifying an agent capable ofinhibiting or stimulating tumor metastasis comprising: (a) preparing asuspension of cells derived from a tumor; (b) introducing a sample ofthe suspension into a first fluid chamber of a bioreactor, thebioreactor, comprising a cell-supporting but cell-permeable matrixseparating at least two fluid chambers with fluid flowing therethroughand at least one gas chamber connected to each of the fluid chambers;(c) supplying to said at least two fluid chambers fluid culture mediaand gas composition suitable to support tumor growth; (d) incubating thebioreactor under conditions and for a time sufficient for cellproliferation and production of metastatic cells; (e) introducing acandidate agent into the first or the second chamber; (f) collectingcells that have migrated into the second chamber; (g) identifying andmonitoring the fraction of metastatic tumor cells among the cellscollected in step (f).
 20. The method of claim 19, wherein the agent isselected from a small-molecule compound, an antibody or a biologic. 21.The method of claim 19, wherein conditions including one or more ofglucose concentration, lactic acid concentration and pH are monitored inthe bioreactor during steps (d)-(e).
 22. The method of claim 19 whereinthe metastatic cells are identified by the presence of one or more ofthe following biomarkers: EpCAM, CK5, CK7, CK18, CK19, Cd44v6, EphB4,FAP (seprase), IGF-1R, BCL2, HER2, CA19-9, CEA, CD133, MUC1, N-cadherin,Survivin and PTEN.
 23. A method of assessing metastatic potential of atumor comprising: (a) preparing a suspension of cells derived from thetumor; (b) introducing a sample of the suspension into a first fluidchamber of a bioreactor, the bioreactor, comprising a cell-supportingbut cell-permeable matrix separating at least two fluid chambers withfluid flowing therethrough and at least one gas chamber connected toeach of the fluid chambers; (c) supplying fluid culture media and gascomposition supportive of tumor growth to said at least two fluidchambers; (d) incubating the bioreactor under conditions and for a timesufficient for cell proliferation and production of metastatic cells;(e) collecting cells that have migrated into the second chamber; (f)identifying metastatic tumor cells among the cells collected in step(e).
 24. The method of claim 23, further comprising assessing thefraction of metastatic tumor cells among the cells collected in step(e).
 25. The method of claim 23, wherein the metastatic cells in step(f) are identified by the presence of one or more of the followingbiomarkers: EpCAM, CK5, CK7, CK18, CK19, Cd44v6, EphB4, FAP (seprase),IGF-1R, BCL2, HER2, CA19-9, CEA, CD133, MUC1, N-cadherin, Survivin andPTEN.
 26. The method of claim 23, wherein conditions including one ormore of glucose concentration, lactic acid concentration and pH aremonitored in the bioreactor during steps (d).